The sphingolipid ceramide promotes cell cycle arrest, differentiation, senescence, and death. The mechanisms by which it affects these cellular processes are poorly defined. My lab has recently published data supporting a new model for ceramide action: starving cells to death. We found that ceramide generation results in rapid and profound nutrient transporter down-regulation in mammalian cells similar to what has been observed in yeast. To respond to this intracellular nutrient limitation, ceramide-exposed cells engage in protective autophagy, a starvation response by which cells digest their constituent molecules to obtain energy and essential nutrients. In further support of this bioenergetic model for ceramide action, supplying cells with a transporter-independent, cell-permeable nutrient, methyl pyruvate, blocked ceramide-induced death. Furthermore, inducing metabolic quiescence by gradually adapting cells to tolerate low extracellular nutrient levels completely eliminated ceramide toxicity. We propose to extend these studies through the following Specific Aims: 1) Identify the molecular pathways between ceramide and nutrient transporter proteins. The mechanisms by which ceramide causes nutrient transporter loss are undefined. We will determine the trafficking step affected by ceramide and whether several established ceramide effector proteins contribute to transporter loss. 2) Determine the mechanism of action for the anti-neoplastic dose of the sphingolipid analog, FTY720. We will determine whether FTY720 kills cells by down-regulating nutrient transporter proteins. We will also test our hypothesis that FTY720 has secondary effects on endocytic trafficking that increase its toxicity. These studies will allow us to refine our bioenergetic model for ceramide action and increase our understanding of how endocytic traffic is regulated. Studies with FTY720 may also serve as proof of the principle that targeting nutrient transporter proteins is a safe and effective therapeutic approach. Ceramide- induced nutrient transporter down-regulation may play an important role in the pathogenesis of cancer and type 2 diabetes. If so, these studies may also identify novel chemotherapeutic targets to treat these prevalent human diseases. PUBLIC HEALTH RELEVANCE: The proposed studies are of broad medical relevance as ceramide plays a key role in many human diseases as well as in aging. The planned studies have particular relevance to cancer and type 2 diabetes and may lead to new therapeutic approaches to these prevalent diseases.